DESCRIPTION: The overall goal of this application is to demonstrate the feasibility of using a microengineered `Lung-on-a-Chip microfluidic device to probe the molecular mechanism of mechano-chemical signaling in the human lung, and to use this knowledge to develop new and improved inhibitors of pulmonary edema development. One of the most rapid (< 5 msec) mechanical signaling events triggered by force transmission from the microenvironment to the cell via their extracellular matrix adhesions involves integrin-dependent activation of the stress-activated membrane ion channel TRPV4, which appears to be critical for the development of many disease processes, including pulmonary edema. The molecular mechanism by which forces applied to integrin mediate this `early- immediate' mechanical signaling response that activates TRPV4 and lead to pulmonary disease is not well understood. To study this process in vitro, we will use a recently developed human Lung-on-a-Chip microfluidic device that contains an artificial alveolar-capillary interface lined by living human lung alveolar and capillary cells hat experiences physiological breathing motions and regenerates a functional vascular permeability barrier in vitro. Importantly, we previously used this microengineered lung chip to show that a specific chemical inhibitor of TRPV4 activity can prevent pulmonary vascular leakage induced by both interleukin-2 and mechanical deformation (breathing motions). In addition, our preliminary results have revealed that the transmembrane protein CD98 binds to both 1-integrin and TRPV4, and that it is required for mechanical, but not chemical, activation of TRPV4. Thus, in this project, we propose to use our microengineered human Lung- on-a-Chip device to delineate the molecular mechanism by which forces applied to integrins activate TRPV4, and to develop new therapeutics for pulmonary edema that targets this molecular mechanism. The specific aims include: 1) to define the molecular mechanism by which CD98 mediates 1-integrin-dependent mechanical activation of TRPV4 in human microvascular endothelial cells, 2) to develop peptide modulators of mechanical signaling through TRPV4 that prevent vascular leakage in the lung-on-a-chip pulmonary edema model, and 3) to validate the peptide inhibitors by demonstrating their ability to prevent vascular leakage in an ex vivo mouse pulmonary edema model.